Myocardial transcription factors are modulated during

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Azakie Fineman He Surgery for Congenital Heart Disease. pression and cardiac hypertrophy 4 12 14 Class I HDACs. Abbreviations and Acronyms may act as prohypertrophic agonists 15 17 Thus isoform. ADP adenosine diphosphate specific inhibitors of class I HDACs and or cardiac specific. ChIP chromatin immunoprecipitation HATs may have an important and specific inhibitory effect. cTnT cardiac troponin T on the development of pathologic cardiac hypertrophy con. HAT histone acetyl transferase,tractile dysfunction and heart failure 15 19. HDAC histone deacetylase,MEF2 myocyte enhancer factor 2. Recently Kee and colleagues15 have shown that inhibi. PAR poly adenosine diphosphate ribose tion of histone deacetylation blocks cardiac hypertrophy. PARP poly adenosine diphosphate ribose induced by angiotensin II infusion and aortic banding Sim. polymerase ilarly Kong and colleagues20 reported that suppression of. TEF 1 transcriptional enhancer factor 1 class I and II HDACs blunts pressure overload cardiac. hypertrophy Suppression of ventricular growth was well. tolerated in terms of both clinical outcome and cardiac. performance measures Interstitial fibrosis was diminished. Various pathologic signals stressors or biomechanical in hearts treated with HDAC inhibitors and collagen syn. forces elicit different intracellular signaling pathways that thesis in isolated cardiac fibroblasts was suppressed There. ultimately stimulate fetal cardiac gene expression in chil was no evidence of cell death or apoptosis Systolic function. dren and adults and pathologic hypertrophy an early mile in the setting of blunted hypertrophic growth was preserved. stone in heart failure 1 2 Although numerous biomechanical as shown by echocardiography and hemodynamic data The. forces and pathologic conditions can result in myocardial hypertrophy associated switch of adult and fetal sarcomeric. dysfunction it is hypothesized that there is a final common isoforms was attenuated which likely contributed to the. pathway in which transcriptional alterations in sarcomeric observed preservation of systolic function in HDAC. gene expression common to many forms of pathologic inhibitor treated hearts Both these studies suggest that. hypertrophy concentric eccentric result in contractile HDAC inhibition is a viable therapeutic strategy that holds. dysfunction of the heart Thus contractile dysfunction of promise in the treatment of pressure load induced heart. the heart can be viewed as a transcriptional disorder 3 4 disease The effects of HDAC inhibition on volume loaded. Transcription factors are nuclear proteins that bind pro heart dysfunction are not known. moter regulatory elements to activate or repress gene ex The aims of the current study are 1 to biochemically and. pression The combinatorial interactions between transcrip morphologically characterize a clinically relevant model of. tion factors and promoter elements that are required for the pathologic hypertrophy and congestive heart failure and 2 to. regulation of cardiac gene expression during normal cardiac determine the changes in expression levels and posttransla. development probably operate during pathologic cardiac tional modifications of selected cardiac transcription factors In. remodeling and hypertrophy NKX2 5 myocyte enhancer utero placement of aorta pulmonary arterial shunts has been. factor 2 MEF2 HAND and GATA transcription factors reported as a model of postnatal pulmonary hypertension with. have been implicated in transcriptional control pattern for increased pulmonary blood flow in lambs21 and is used in this. mation and segmental control of cardiac development 5 8 study as a model of pathologic cardiac hypertrophy Surgically. Using the cardiac troponin T cTnT promoter as a model created aorta pulmonary shunts in fetal lambs produced left. gene we have shown that transcriptional enhancer factor 1 ventricular volume overload and right ventricular pressure. TEF 1 MEF2 and Sp family of transcription factors also overload leading to altered contractile protein gene expression. contribute to the developmental regulation of cardiac gene biventricular hypertrophy and congestive heart failure In the. expression in avian embryonic cardiomyocytes 9 10 These shunted lamb model pathologic cardiac remodeling and dele. myocardial transcription factors which are important for terious cardiac gene expression occurred in association with. normal growth development and maintenance of the car changes in the levels and modification states of cardiac specific. diac phenotype may also be implicated during induction of transcription factors GATA 4 DTEF 1 Sp1 Sp3 and. fetal genes and the upregulation of constitutive genes that is NKX2 5 Furthermore we show that functionally TEF 1. seen with pathophysiologic hypertrophy 2 11 MEF2 and Sp1 factors which bind the cTnT promoter within. Recent interest in transcriptional disorders of the heart the context of chromatin transactivate the cTnT promoter in. has also implicated cardiac histone acetylation in the devel ovine cardiomyocytes implicating them as important regula. opment of pathologic cardiac hypertrophy and heart failure tors of the pathologic hypertrophic process. Histone acetyl transferases HATs are believed to acetylate. histone proteins relax chromatin and expose prohypertro Materials and Methods. phic genes for activation by cardiogenic transcription fac Reagents. tors 12 13 Histone deacetylases HDAC classes I and II have Media serum enzymes reagents and materials for tissue culture. also been implicated in regulation of myocardial gene ex were purchased from the Cell Culture facility at the University of. The Journal of Thoracic and Cardiovascular Surgery Volume 132 Number 6 1263. Surgery for Congenital Heart Disease Azakie Fineman He. California San Francisco Oligonucleotides were purchased from Surgical Preparations and Care Ewes. Operon Technologies Inc Alameda Calif or Invitrogen Carlsbad Pregnant mixed breed Western ewes 135 140 days gestation. Calif term 145 days were operated on under sterile conditions. Through a left lateral fetal thoracotomy an 8 0 mm polytetrafluo. roethylene vascular graft 2 mm length W L Gore Associates. Plasmids and Constructs Tissue Culture Cell Inc Flagstaff Ariz was sewn between the ascending aorta and. Transfections and Reporter Gene Assays main pulmonary artery All lambs were spontaneously delivered. The promoter reporter constructs used in this study consist of and were instrumented as previously described 21. 268 nucleotides of the chicken cTnT promoter upstream of the. transcription initiation site Gallus domesticus cardiac isoform Heart Tissue Preparation. of troponin T TNT gene exon 1 and promoter sequence Whole heart and chamber specific weights were measured in all. ACCESSION M5790 Luciferase reporter constructs were cre shunted and control specimens Samples of right atrium right. ated and subcloned into the plasmid pGL2 Promega Madison ventricle left atrium left ventricle and ventricular septum were. Wis as previously described 9 excised weighed and used to make fresh nuclear preparations. Hearts were harvested from neonatal lambs and cardiomyo Whole tissue and excess nuclear preparations were snap frozen in. cyte cultures established by standard techniques 10 The differ liquid nitrogen Samples were stored at 70 C until used for. entiation state of cultured cardiomyocytes was determined by analysis. light microscopy presence of beating cardiomyocytes immu. nocytochemistry for MF20 and Western blotting for myosins Determination of DNA Content. cTnT GATA 4 MEF2 and NKX2 5 Plasmid constructs were DNA content was determined by standard methods Genomic. transfected according to the manufacturer s protocol Effectene DNA mini prep Bay Gene Inc San Francisco Calif Values were. Transfection kit Qiagen Valencia Calif with minor modifi normalized to gram of cardiac muscle. cations 9 Reporter gene activity was determined with the Dual. Luciferase reporter assay DLR Promega Madison Wis Fire Statistical Analysis. fly light intensity was read on a Luminometer TD 20 20 DLR Comparisons between shunt and age matched controls were made. Ready Turner Designs Instrument Sunnyvale Calif Cotrans by an unpaired t test. fection with 20 ng phRL TK reporter was used to standardize All protocols and procedures were approved by the Committee. for transfection variability Cotransfection efficiency of Sp on Animal Research of the University of California San Fran. MEF2 and DTEF 1 expression constructs was verified with cisco All animals were put to death by appropriate methods as. pSV galactosidase Promega Madison Wis and or pAAV described in the National Institutes of Health Guidelines for the. GFP staining Reporter gene activity was expressed as mean Care and Use of Laboratory Animals. standard deviation Statistical comparisons were performed by. paired t tests Results,In Utero Placement of Aorta Pulmonary Shunts. Nuclear Extracts Results in Growth Failure Four chamber. Cardiac muscle was harvested from 2 week old lambs control and Cardiomegaly and Biventricular Hypertrophy. shunted at 4 C Nuclear extracts were prepared by standard meth Shunted lambs showed clinical evidence of failure to thrive. ods Active Motif Inc Carlsbad Calif Compared with control animals shunted lambs were tachy. pneic and demonstrated less activity and lower tolerance to. Western Blot Analysis and Densitometry stress after 1 week of age By 2 weeks of age shunted lambs. Total protein concentration was quantitated with the Bio Rad DC had significantly slower weight gain than controls The. protein assay kit Bio Rad Laboratories Hercules Calif An equal hearts of shunted lambs were significantly larger than those. amount of protein was loaded in each lane for Western blots of controls Figure 1 Table 1 and Figure E1 All four. sodium dodecylsulfate polyacrylamide gel electrophoresis was chambers showed substantial increases in mass without. used to separate nuclear proteins 10 g on a 10 gel and significant increases in DNA content suggesting that hy. followed by transfer to a polyvinylidene difluoride membrane. pertrophy and not hyperplasia was the predominant stress. Amersham Biosciences GE Healthcare Bio Sciences Corp Pis. response The left ventricle was dilated and showed evi. cataway NJ Membranes were then blocked and incubated with. antibody as previously described 9 Reactive bands were visualized. dence of eccentric hypertrophy while the right ventricle. with the SuperSignal West Femto Maximum Sensitivity Substrate was predominantly thick walled with evidence of concen. Kit Pierce Rockford Ill and Kodak 440CF image station tric hypertrophy. Kodak New Haven Conn The image was selected and analyzed. with the public domain program NIH Image and band intensity Hemodynamics of 2 week old Shunted Lambs. was quantified Parallel sodium dodecylsulfate gels and immuno Table 1 summarizes the hemodynamics of shunted versus. blots of actin and glyceraldehyde 3 phosphate dehydrogenase control lambs at 2 weeks of age Shunted animals had. controls were performed to verify sample integrity and loaded significantly higher mean pulmonary artery pressures with a. quantity mean pulmonary systemic flow ratio of 2 9 1 Left and right. 1264 The Journal of Thoracic and Cardiovascular Surgery December 2006. Azakie Fineman He Surgery for Congenital Heart Disease. Figure 1 Gross pathology of shunted hearts at 2 weeks of age. Left panel Photograph of shunted heart Right panel Photograph. of control heart, atrial pressures were significantly higher in shunted. Sarcomeric Protein and Myocardial Transcription,Factor Levels Are Altered During Pathophysiologic.
Shunting in the Neonatal Ovine Heart, Western blots of shunted n 5 and control n 5 hearts. were analyzed by band densitometry The hearts of 2 week. old shunted lambs showed significant increases in myosin. and cTnT expression Figure 2 and Figure 3 A and B, Fetal high molecular weight isoforms of cTnT were up. regulated consistent with the activation of a pathologic. hypertrophic program Figure 2 The increase in sarco Figure 2 Characteristic Western blot of sarcomeric proteins and. selected transcription factors in 2 week old control and shunted. meric protein expression and associated fetal isoforms was. lambs Antibody used for Western blot analysis is listed on left MF20. seen in all four chambers Associated with the increase in antimyosin and cTnT levels are increased in shunted animals. sarcomeric proteins a variety of cardiac transcription fac consistent with the hypertrophy and increased mass seen on gross. tors known to govern cardiac gene expression during em pathologic examination Fetal embryonic isoforms of cTnT are ex. pressed in the hypertrophied shunted hearts There are associated. increases in DTEF 1 MEF2 not shown GATA4 NKX2 5 and Sp1. TABLE 1 General hemodynamics in 2 week old lambs levels and concomitant decreases in Sp3 levels within the cardiac. Control Shunted chambers see Figure 5 actin and glyceraldehyde 3 phosphate. lambs lambs dehydrogenase GAPDH control proteins are comparable in shunted. and control specimens RA Right atrium RV right ventricle LA left. Mean arterial blood pressure mm Hg 65 7 55 7, atrium LV left ventricle Sept interventricular septal muscle MW. Pulmonary arterial pressure mm Hg 15 2 22 4,kDa molecular weight in kilo Daltons. Left pulmonary blood flow 60 21 157 28,mL kg 1 min 1.
Heart rate beats min 171 24 174 25,Left atrial pressure mm Hg 3 1 7 7 3 4. cardiac transcription factors in the regulation of cardiac gene expression during pathologic hypertrophy in vivo The cardiac troponin T promoter is used as a model gene Methods and Results Transient transfections of ovine cardiomyocytes in culture show that Sp1 transcriptional enhancer factor 1 and myocyte enhancer factor 2

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